PL9008: Key publications on the characterization of gene vectors by light scattering

Significance of the Topic

Light scattering–based hyphenated techniques are indispensable for reliable characterization of gene delivery nanoparticles. They deliver rapid, label-free insights into particle size, heterogeneity, composition and aggregation. This information underpins quality control, process development and regulatory compliance in gene therapy, vaccine production and nanomedicine formulation.

Objectives and Overview

This collection of key publications demonstrates how high-throughput dynamic light scattering (HT-DLS), size-exclusion chromatography with multi-angle light scattering (SEC-MALS), ion-exchange chromatography with MALS (IEX-MALS) and asymmetrical-flow field-flow fractionation with MALS (FFF-MALS) have been applied to characterize viral vectors, lipid nanoparticles, polymeric carriers and extracellular vesicles. The primary goals include quantifying particle size distribution, determining cargo loading (e.g., capsid content, RNA), and optimizing formulation processes.

Methodology and Instrumentation

All studies combine light scattering detectors with separation or high-throughput platforms:

• HT-DLS platforms for rapid size profiling of formulation libraries.
• SEC-MALS and IEX-MALS to resolve size fractions and measure molecular weight and particle concentration.
• FFF-MALS for separation of particle subpopulations based on hydrodynamic size without a stationary phase.
• Dynamic/static light scattering (DLS/SLS) for complementary size and surface charge measurements.

Key Results and Discussion

Viral vectors studies established robust methods for capsid fill-state quantification and size distribution of AAV and VLPs. SEC-MALS and IEX-MALS enabled differentiation of empty and full capsids.
FFF-MALS facilitated single-particle analysis of virions and assessment of assembly efficiency.
Lipid nanoparticle research linked particle size to immunogenicity, provided high-throughput screening of oligonucleotide-loaded LNPs, and demonstrated online determination of RNA loading.
Polymeric nanoparticle work used SEC-MALS to determine polymer molecular weight and correlate it with siRNA delivery efficacy.
Extracellular vesicle investigations applied HT-DLS for plasma exosome profiling and FFF-MALS for separation of exomeres and small EV subtypes, revealing distinct biophysical signatures.

Benefits and Practical Applications

• Non-invasive, label-free quantification of particle size, mass and concentration.
• High throughput screening expedites formulation development.
• Separation-coupled detection resolves complex mixtures into subpopulations.
• Real-time monitoring supports process analytical technology (PAT) in biomanufacturing.

Future Trends and Possibilities

Integration of microfluidic fractionation, real-time in-line detectors, and machine-learning algorithms promises more predictive and automated nanoparticle characterization. Standardized protocols for regulatory approval will emerge. Advanced multi-detector platforms may expand to novel carriers such as exomeres and multifunctional nanomedicines.

Conclusion

Light scattering–based hyphenated techniques are central to the analytical toolbox for gene delivery systems. They offer quantitative, high-resolution insights that drive formulation optimization, quality control, and innovation in nanomedicine.

Reference

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